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Biophase concentrations of antiepileptic drugs can differ significantly from pharmacokinetics in plasma. A crucial determinant in the disposition of antiepileptic drugs to the brain is represented by the blood-brain barrier. There is growing evidence that this barrier can alter the availability of antiepileptic drugs at the target site. The permeability of the blood-brain barrier becomes particularly relevant in epileptic conditions and in drug refractory situations. In vivo, intracerebral microdialysis is a valuable technique to determine biophase drug concentrations as it enables investigation of antiepileptic drug transport and distribution in the brain as a function of time. The present review illustrates that intracerebral microdialysis is an indispensable tool for the assessment of the pharmacokinetics of antiepileptic drugs. In addition, we demonstrate how microdialysis data can be used in conjunction with mechanism-based pharmacokinetic/pharmacodynamic modeling for dose selection and optimization of the therapeutic regimen for novel compounds.

Several types of brain injuries have been associated with alterations in the striatal expression of neurotrophic factors, including glial cell line-derived neurotrophic factor (GDNF). However, contradictory results on the striatal expression of GDNF have been reported in different animal models of Parkinson's disease. For this reason, we examined the effect of nigrostriatal damage on the mRNA and protein expression levels of GDNF in the striatum as a function of time following a striatal or medial forebrain bundle 6-hydroxydopamine lesion. At different time points after the administration of 6-hydroxydopamine, striatal expression levels of GDNF were analyzed with semi-quantitative Western blotting. No significant changes in GDNF expression levels were observed within the 35-day observation period, either between the denervated and the intact striatum of medial forebrain bundle and striatally lesioned rats or between the striata of lesioned animals and those of control animals. In order to reinforce these results, striata of lesioned rats, sacrificed 18 days after lesioning, were analyzed with enzyme-linked immunosorbent assay and real-time polymerase chain reaction. At this time point, both techniques confirmed the results of the Western blot analysis, detecting no changes in striatal expression of GDNF, either at the protein level, or at the mRNA level. These data show that nigrostriatal damage induced by 6-hydroxydopamine has no effect on the striatal expression of GDNF.

Liquid chromatography coupled to atmospheric pressure ionization tandem mass spectrometry is currently the method of choice for the quantitative determination of drugs in biological matrices. The advantages of this technique include high specificity, sensitivity and throughput. However, co-eluting matrix components, which are not observed in the chromatogram, can have a detrimental effect on the analysis, since they can cause ion suppression or enhancement of the analyte. The evaluation of matrix effects on the quantitative analysis of drugs in biological fluids is an important and sometimes overlooked aspect of assay validation. In this review, the influence of matrix effects on bioanalytical LC-MS/MS methods is discussed and illustrated with some examples. In addition, possible solutions to reduce or eliminate matrix effects are highlighted. A literature overview of validated LC-MS/MS methods published from January till June 2008 is also included. Although matrix effects are investigated in most papers, there is no consensus on how matrix effects should be evaluated during method validation. In addition, the definition of specificity should be changed for LC-MS/MS based methods.

Glutamate, the most abundant excitatory neurotransmitter in the central nervous system, is well known to be implicated in epileptic seizures. Therefore, impairments in glutamate transport could have an involvement in the mechanism of epileptogenesis. The uptake of glutamate into synaptic vesicles is mediated by vesicular glutamate transporters (vGLUTs). There are three known vGLUT isoforms, vGLUT1-3. In this study, we are particularly interested in the vGLUT2 isoform. We investigated the possible role of vGLUT2 in pentylenetetrazol (PTZ)-induced seizure generation. Seizure threshold of PTZ was compared in vGLUT2 heterozygous knock out (HET) and wild type (WT) mice. In comparison with their WT littermates a lower dose of PTZ was needed in the vGLUT2 HET mice until the onset of the first myoclonic jerk. The threshold for PTZ-induced clonic seizure activity was also lower in the vGLUT2 HET mice. These results indicate, for the first time, that vGLUT2 is likely involved in the epileptogenesis of generalized seizures.

The IRAP ligands Angiotensin IV (Ang IV) and LVV-haemorphin 7 (LVV-H7) enhance performance in a range of memory paradigms in normal rats and ameliorate memory deficits in rat models for amnesia. The mechanism by which these peptides facilitate memory remains to be elucidated. In recent in vitro experiments, we demonstrated that Ang IV and LVV-H7 potentiate activity-evoked glucose uptake into hippocampal neurons. This raises the possibility that IRAP ligands may facilitate memory in hippocampus-dependent tasks through enhancement of hippocampal glucose uptake. Acute intracerebroventricular (i.c.v.) administration of 1nmol Ang IV or 0.1nmol LVV-H7 in 3 months-old Sprague-Dawley rats enhanced spatial working memory in the plus maze spontaneous alternation task. Extracellular hippocampal glucose levels were monitored before, during and after behavioral testing using in vivo microdialysis. Extracellular hippocampal glucose levels decreased significantly to about 70% of baseline when the animals explored the plus maze, but remained constant when the animals were placed into a novel control chamber. Ang IV and LVV-H7 did not significantly alter hippocampal glucose levels compared to control animals in the plus maze or control chamber. Both peptides had no effect on hippocampal blood flow as determined by laser Doppler flowmetry, excluding that either peptide increased the hippocampal supply of glucose. We demonstrated for the first time that Ang IV and LVV-H7 enhance spatial working memory in the plus maze spontaneous alternation task but no in vivo evidence was found for enhanced hippocampal glucose uptake or blood flow.

Background: Bupivacaine has a lower incidence of transient neurological symptoms than lidocaine after intrathecal (i.t.) injection. The increased toxic potential of lidocaine does not support its use in the clinical setting and could be related to augmented levels of spinal prostaglandin E(2) (PGE(2)). We tested whether levobupivacaine leads to lower PGE(2) levels than lidocaine. Moreover, we compared the release of PGE(2) and glutamate after i.t. injections of levobupivacaine or lidocaine., Methods: Rats were anaesthetized for implantation of an i.t. dialysis catheter. This allowed sampling dialysates of cerebrospinal fluid (CSF) for measuring PGE(2) and glutamate levels. The microdialysis setting included baseline sampling and was followed by an i.t. injection of levobupivacaine 250 microg, 100 microg, or saline. PGE(2) and glutamate levels in CSF were analysed for 4 h. In addition, the residual effect of a second i.t. injection on, respectively, of PGE(2) and glutamate changes was compared after injection of either 250 or 100 microg levobupivacaine, 1000 or 400 microg lidocaine, or saline., Results: Prolonged spinal PGE(2) increases lasting 50-120 min were observed after levobupivacaine injection. Higher PGE(2) concentrations were observed after the second lidocaine 1000 microg injection. Glutamate release after the second injection did not vary between the local anaesthetic groups., Conclusions: Spinal PGE(2) levels are similarly increased after i.t. levobupivacaine injection of 250 and 100 microg. A higher PGE(2) response was observed after a second i.t. injection in the animals receiving 1000 microg lidocaine than those receiving 400 mg lidocaine or either dose of levobupivacaine.

Background and Purpose: Accurate prediction of biophase pharmacokinetics (PK) is essential to optimize pharmacotherapy in epilepsy. Here, we characterized the PK of the active metabolite of oxcarbazepine, 10,11-dihydro-10-hydroxy-carbamazepine (MHD) in plasma and in the hippocampus. Simultaneously, the impact of acute seizures and efflux transport mechanisms on brain distribution was quantified., Experimental Approach: Rats received subtherapeutic and anticonvulsant doses of MHD in non-epileptic conditions and during focal pilocarpine-induced limbic seizures. To evaluate the effect of efflux transport blockade, a separate group received subtherapeutic doses combined with intrahippocampal perfusion of verapamil. Free plasma and extracellular hippocampal MHD concentrations were determined using microdialysis and liquid chromatography techniques. An integrated PK model describing simultaneously the PK of MHD in plasma and brain was developed using nonlinear mixed effects modelling. A bootstrap procedure and a visual predictive check were performed to assess model performance., Key Results: A compartmental model with combined zero- and first-order absorption, including lag time and biophase distribution best described the PK of MHD. A distributional process appeared to underlie the increased brain MHD concentrations observed following seizure activity and efflux transport inhibition, as reflected by changes in the volume of distribution of the biophase compartment. In contrast, no changes were observed in plasma PK., Conclusions and Implications: Simultaneous PK modelling of plasma and brain concentrations has not been used previously in the evaluation of antiepileptic drugs (AEDs). Characterisation of biophase PK is critical to assess the impact of efflux transport mechanisms and acute seizures on brain disposition and, consequently, on AED effects.

The central angiotensin system plays a crucial role in cardiovascular regulation. More recently, angiotensin peptides have been implicated in stress, anxiety, depression, cognition, and epilepsy. Angiotensin II (Ang II) exerts its actions through AT(1) and AT(2) receptors, while most actions of its metabolite Ang IV were believed to be independent of AT(1) or AT(2) receptor activation. A specific binding site with high affinity for Ang IV was discovered and denominated "AT(4) receptor". The beneficiary effects of AT(4) ligands in animal models for cognitive impairment and epileptic seizures initiated the search for their mechanism of action. This proved to be a challenging task, and after 20 years of research, the nature of the "AT(4) receptor" remains controversial. Insulin-regulated aminopeptidase (IRAP) was first identified as the high-affinity binding site for AT(4) ligands. Recently, the hepatocyte growth factor receptor c-MET was also proposed as a receptor for AT(4) ligands. The present review focuses on the effects of Ang II and Ang IV on synaptic transmission and plasticity, learning, memory, and epileptic seizure activity. Possible interactions of Ang IV with the classical AT(1) and AT(2) receptor subtypes are evaluated, and other potential mechanisms by which AT(4) ligands may exert their effects are discussed. Identification of these mechanisms may provide a valuable target in the development in novel drugs for the treatment of cognitive disorders and epilepsy.

Excitatory amino acid transporter 4 (EAAT4), a member of the high-affinity Na+/K+-dependent glutamate transporter family, is highly enriched in Purkinje cells of the cerebellum, although it is not restricted to these cells. The detailed expression of EAAT4 protein in different adult rat fore- and midbrain regions was examined. Despite moderate expression levels compared with the cerebellum, EAAT4 protein was omnipresent throughout the fore- and midbrain. With antibodies raised against the N-terminal mouse EAAT4 sequence, the highest protein expression levels were observed in the substantia nigra pars compacta, ventral tegmental area, paranigral nucleus, habenulo-interpeduncular system, supraoptic nucleus, lateral posterior thalamic nucleus, subiculum, and superficial layers of the superior colliculus. Relatively high levels of EAAT4 protein were also detected in the hippocampal principal cells, in the glutamatergic, gamma-aminobutyric acid (GABA)ergic, dopaminergic and most likely cholinergic cells of all nuclei of the basal ganglia, and in neurons of layers II/III and V of the cerebral cortex. The expression of EAAT4 was confirmed at the mRNA level in some important fore- and midbrain structures by in situ hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) and estimated to range from 6.7 to 1.6% of the amount in the cerebellum as measured by real-time PCR., ((c) 2008 Wiley-Liss, Inc.)

Background: In this study, we have investigated whether intrathecal (i.t.) lidocaine administration is accompanied with changes of cerebrospinal fluid (CSF) prostaglandin E(2) (PGE(2)) levels., Methods: Rats were anaesthetized for i.t. implantation of a triple-lumen spinal loop dialysis catheter. CSF changes in PGE(2) after i.t. injection of saline, 400, or 1000 microg of lidocaine were measured. The impact of i.t. pretreatment with 5 microg MK801 (N-methyl-D-aspartate glutamate antagonist) or 10 microg SC76309A (COX-2 inhibitor) was also investigated. CSF dialysates for measurement of PGE(2) were collected for 4 h. During the whole procedure, motor and sensory blocks were evaluated. A separate group receiving i.t. lidocaine 400 microg (without dialysate sampling) was assessed for mechanical (Von Frey) and radiant heat pain., Results: PGE(2) levels increased to 400% of baseline and remained elevated for 90-120 min after i.t. lidocaine at both doses. Pretreatment with SC76309A and MK801 attenuated this increase. A 40 min period of enhanced pain response was observed after Von Frey filament stimulation during and after sensory and motor block recovery., Conclusions: I.T. lidocaine (400 or 1000 microg) increases PGE(2) levels in the CSF for 90-120 min along with a transient period of mechanical hyperalgesia after sensory and motor block recovery.

Altered glutamate signaling is associated with Parkinson's disease. To study the involvement of the cystine/glutamate antiporter in the pathogenesis of Parkinson's disease, we developed new polyclonal antibodies recognizing xCT, the specific subunit of this antiporter. The striatal xCT protein expression level was investigated in a hemi-Parkinson rat model, using semiquantitative western blotting. We observed time-dependent changes after a unilateral 6-hydroxydopamine lesion of the nigrostriatal pathway with increased expression levels in the deafferented striatum after 3 weeks. Twelve weeks postlesion, expression levels returned to normal. These data suggest, for the first time, an involvement of the cystine/glutamate antiporter in determining the aberrant glutamate neurotransmission in the striatum of a parkinsonian brain.

For decades, angiotensin (Ang) II was considered as the end product and the only bioactive peptide of the renin-angiotensin system (RAS). However, later studies revealed biological activity for other Ang fragments. Amongst those, Ang IV has drawn a lot of attention since it exerts a wide range of central and peripheral effects including the ability to enhance learning and memory recall, anticonvulsant and anti-epileptogenic properties, protection against cerebral ischemia, activity at the vascular level and an involvement in atherogenesis. Some of these effects are AT(1) receptor dependent but others most likely result from the binding of Ang IV to insulin-regulated aminopeptidase (IRAP) although the exact mechanism(s) of action that mediate the Ang IV-induced effects following this binding are until now not fully known. Nevertheless, three hypotheses have been put forward: since Ang IV is an inhibitor of the catalytic activity of IRAP, its in vivo effects might result from a build-up of IRAP's neuropeptide substrates. Second, IRAP is co-localized with the glucose transporter GLUT4 in several tissue types and therefore, Ang IV might interact with the uptake of glucose. A final and more intriguing hypothesis ascribes a receptor function to IRAP and hence an agonist role to Ang IV. Taken together, it is clear that further work is required to clarify the mechanism of action of Ang IV. On the other hand, a wide range of studies have made it clear that IRAP might become an important target for drug development against different pathologies such as Alzheimer's disease, epilepsy and ischemia.

Liquid chromatography with amperometric detection remains the most widely used method for acetylcholine quantification in microdialysis samples. Separation of acetylcholine from choline and other matrix components on a microbore chromatographic column (1 mm internal diameter), conversion of acetylcholine in an immobilized enzyme reactor and detection of the produced hydrogen peroxide on a horseradish peroxidase redox polymer coated glassy carbon electrode, achieves sufficient sensitivity for acetylcholine quantification in rat brain microdialysates. However, a thourough validation within the concentration range required for this application has not been carried out before. Furthermore, a rapid degradation of the chromatographic columns and enzyme systems have been reported. In the present study an ion-pair liquid chromatography assay with amperometric detection was validated and its long-term stability evaluated. Working at pH 6.5 dramatically increased chromatographic stability without a loss in sensitivity compared to higher pH values. The lower limit of quantification of the method was 0.3 nM. At this concentration the repeatability was 15.7%, the inter-day precision 8.7% and the accuracy 103.6%. The chromatographic column was stable over 4 months, the immobilized enzyme reactor up to 2-3 months and the enzyme coating of the amperometric detector up to 1-2 months. The concentration of acetylcholine in 30 μl microdialysates obtained under basal conditions from the hippocampus of freely moving rats was 0.40 ± 0.12 nM (mean ± SD, n = 30). The present method is therefore suitable for acetylcholine determination in rat brain microdialysates.

Background and Purpose: Insulin-regulated aminopeptidase (IRAP) and the insulin-dependent glucose transporter GLUT4 colocalize in specific intracellular vesicles (that is, GLUT4 vesicles). These vesicles move slowly to the cell surface, but their translocation is markedly enhanced by insulin, resulting in higher glucose uptake. Previous studies of the insulin-mediated translocation of IRAP to the cell surface have been hampered by the laborious detection of IRAP at the cell surface. We aimed to develop a more direct and faster method to detect IRAP. To this end, we used model systems with well-characterized IRAP: CHO-K1 cells expressing endogenous IRAP and recombinant HEK293 cells expressing human IRAP. A more widespread application of the method was demonstrated by the use of 3T3-L1 adipocytes., Experimental Approach: After stimulation of the cells with insulin, internalization of IRAP was inhibited by the addition of phenyl arsine oxide (PAO). Then, cell-surface IRAP was detected by the high-affinity binding of radiolabelled angiotensin (Ang) IV (either 125I or 3H)., Key Results: We monitored the time- and concentration dependence of insulin-mediated translocation of IRAP in both cell lines and 3T3-L1 adipocytes. A plateau was reached between 6 and 8 min, and 10(-7) M insulin led to the highest amount of IRAP at the cell surface., Conclusions and Implications: Based on the capacity of the IRAP apoenzyme to display high affinity for radiolabelled Ang IV and on the ability of PAO to inhibit IRAP internalization, we developed a more direct and faster method to measure insulin-mediated translocation of IRAP to the cell surface.

The neurobiological relationships between epilepsy and depression are receiving increased experimental attention. A key role for limbic monoamines in depression has been established and we recently showed the importance of hippocampal monoamines in limbic seizure control. We here studied whether anticonvulsant compounds are antidepressant and can elevate hippocampal dopamine (DA) or serotonin (5-HT) levels determined by in vivo microdialysis in rats. We used assessment of seizure severity in the focal pilocarpine model, antidepressant-like activity within the rat forced swim and the mouse tail suspension tests, and locomotor activity in an open field as behavioural tests. We studied the tricyclic antidepressant imipramine, the selective 5-HT reuptake inhibitor citalopram and the selective DA reuptake blocker GBR-12909. These compounds with combined antidepressant-anticonvulsant properties all directly enhanced extracellular hippocampal DA or 5-HT levels. Since glutamate-mediated hyperexcitability in temporal lobe regions seems to be involved in disturbed emotional behaviour, we next investigated possible antidepressant effects and hippocampal DA or 5-HT modulations exerted by selective ionotropic and metabotropic glutamate receptor ligands with anticonvulsant properties. Combined anticonvulsant-antidepressant activities of the NMDA antagonist MK-801 and the mGluR group I antagonists (AIDA, MPEP) were also associated with locally elicited increases in hippocampal DA and/or 5-HT levels. This study highlights that the hippocampus is an important site of action of combined anticonvulsant-antidepressant and monoamine enhancing effects.

Microdialysis in intact and denervated striatum of unilaterally 6-hydroxydopamine (6-OHDA) lesioned rats was used to investigate whether CR 3394, N-[2-(3,5-dimethyl-1-adamantyl)ethyl]acetamidine, an adamantane derivative with preferential selectivity for the NR2B subunit of the NMDA receptor, has dopamine releasing properties in vivo. We also investigated whether this NMDA antagonist can potentiate the effects of L-Dopa on extracellular dopamine in these animals. After systemic injection, there was no significant effect of CR 3394 on extracellular dopamine, at all doses studied (1, 5 and 20 mg/kg i.p.), in either intact or in denervated striatum. On the other hand, striatal perfusion with 100 microM of the compound elicited release of dopamine in intact, but not in denervated striatum. In denervated striatum of the 6-OHDA-lesioned rats, CR 3394 (5 mg/kg) significantly enhanced the dopamine release induced by L-Dopa administration (25 mg/kg i.p.) in combination with benserazide (10 mg/kg i.p.). In particular, the onset of action of L-Dopa was potentiated. However, when combined with a subthreshold dose of L-Dopa (5 mg/kg), the effects of CR 3394 were lost. We conclude that CR 3394, like other NR2B receptor antagonists, has dopamine releasing properties in vivo. It enhances the effects of suprathreshold doses of L-Dopa in the denervated striatum, but not of low doses of L-Dopa. Therefore, future studies are necessary to establish the potential of selective NR2B receptor antagonists as L-Dopa-sparing agents.

Microdialysis (MD) is an in vivo sampling technique used to investigate biochemical events in the extracellular fluid of animal and human tissues. MD produces protein- and cell-free, aqueous samples which can be analyzed without further sample clean-up. Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a sensitive and selective analysis technique which is suitable to quantify low concentrated target analytes in microdialysates. This paper reviews the LC-MS/MS methods which are described for the quantification of endogenous molecules, such as neurotransmitters and peptides, and of exogenous molecules, such as drugs, in microdialysates. Since miniaturization of the LC-MS/MS methods is the key to obtain maximal sensitivity of the analytical technique, this feature is discussed in the paper. In addition, critical issues related to the quantification of low concentrated molecules in microdialysates are described such as the presence of matrix effects, the low MD efficiency and the sticking of, for instance, neuropeptides.

The purpose of the present study was to identify the effects of an acute injection of a dual dopamine (DA)/noradrenaline (NA) reuptake inhibitor (bupropion) on exercise performance, thermoregulation and neurotransmitters in the preoptic area and anterior hypothalamus (PO/AH) of the rat during exercise in the heat. Body core temperature (T(core)), brain temperature (T(brain)) and tail skin temperature (T(tail)) were measured. A microdialysis probe was inserted in the PO/AH, and samples for measurement of extracellular DA, NA and serotonin (5-HT) levels were collected. Rats received either bupropion (17 mg kg(-1); hot-BUP) or saline (1 ml kg(-1); hot) 20 min before the start of exercise and ran at a speed of 26 m min(-1) until exhaustion in a warm environment (30 degrees C). Rats also ran until exhaustion in a cool environment (18 degrees C; cool). Running time to exhaustion was significantly influenced by the ambient temperature, and it was increased by bupropion in the heat (cool, 143.6 +/- 21 min; hot, 65.8 +/- 13 min; hot-BUP, 86.3 +/- 7.2 min). T(core) and T(brain) at exhaustion were significantly higher in the bupropion group compared to the cool and hot groups, respectively. T(tail) measured at exhaustion was not significantly different between the two hot conditions. Extracellular concentrations of DA and NA in the PO/AH increased during exercise, and was significantly higher in the bupropion than in cool and hot groups (P < 0.05). No differences were observed between groups for 5-HT levels. These results suggest that DA and NA in the PO/AH might be responsible for the increase in exercise performance and T(core) and T(brain) in the bupropion group in hyperthermia. Moreover, these results support previous findings in humans that acute bupropion ingestion increases T(core) during exercise in the heat, indicating the possibility of an important role for DA and NA in thermoregulation.

Increases in hippocampal extracellular neurotransmitter levels have consistently been observed during temporal lobe seizures in humans, but animal studies on this subject have yielded conflicting results. Our aim was to better characterise the relationship between seizure activity and changes in hippocampal glutamate, GABA, dopamine and serotonin by comparing three limbic seizure models which differ only in the pharmacological mechanism used to induce seizures. Seizures were evoked in freely moving rats by intrahippocampal microperfusion, via a microdialysis probe, of the muscarinic receptor agonist pilocarpine (10mM), GABA(A) receptor antagonist picrotoxin (100microM) or group I metabotropic glutamate receptor agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) (1mM). Seizure-related behavioural changes were scored and hippocampal extracellular glutamate, GABA, dopamine and serotonin concentrations were monitored. Seizures were of comparable severity in all groups. During seizures, hippocampal glutamate, GABA and dopamine concentrations increased in all groups. Glutamate increases were significantly higher in the picrotoxin group. Hippocampal serotonin concentration increased following pilocarpine and picrotoxin, but not DHPG. Our results suggest a direct relationship between seizure activity and increased hippocampal extracellular concentrations of glutamate, GABA and dopamine, but not serotonin. The fact that picrotoxin induces seizures by disinhibition, rather than direct excitation, may account for the larger glutamate increases in this group.

The potent anticonvulsant properties of neuropeptide Y (NPY) are generally attributed to a Y2 receptor-mediated inhibition of glutamatergic synaptic transmission. Independent studies have shown that NPY increases brain dopamine content, possibly via interaction with sigma 1 receptors. Recently, we showed that increased extracellular hippocampal dopamine attenuates pilocarpine-induced limbic seizures via activation of hippocampal D2 receptors. Our aim in this study was to elucidate the role of increased hippocampal dopamine in the mechanism of the anticonvulsant action of NPY and to investigate the involvement of Y2 and sigma 1 receptors in this process. Limbic seizures were evoked in freely moving rats by intrahippocampal administration of pilocarpine via a microdialysis probe. NPY was administered intracerebroventricularly, intrahippocampally via the microdialysis probe, or coadministered intrahippocampally with the D2 receptor antagonist remoxipride, the Y2 receptor antagonist BIIE0246 or the sigma 1 receptor antagonist BD1047. Changes in hippocampal extracellular dopamine were monitored, and behavioural changes indicative of seizure activity were scored. Intracerebroventricular (10 nmol/3 microL) and intrahippocampal (20-50 microm) NPY administration increased hippocampal dopamine and attenuated pilocarpine-induced seizures. Hippocampal D2 receptor blockade (4 microm remoxipride) reversed the anticonvulsant effect of NPY. Y2 receptor blockade (1 microm BIIE0246) reversed the anticonvulsant effect of NPY but did not prevent NPY-induced increases in hippocampal dopamine. Sigma 1 receptor blockade (10 microm BD1047) abolished NPY-induced increases in hippocampal dopamine and reversed the anticonvulsant effect of NPY. Our results indicate that NPY-induced increases in hippocampal dopamine are mediated via sigma 1 receptors and contribute to the anticonvulsant effect of NPY via increased activation of hippocampal D2 receptors. This novel mechanism of anticonvulsant action of NPY is separate from, and may be complementary to, the well established Y2 receptor-mediated inhibition of hippocampal excitability.

The present study investigated the effects of N-methyl-D-aspartic acid.H2O (NMDA) on the dopamine, glutamate and GABA release in the subthalamic nucleus (STN) by using in vivo microdialysis in rats. NMDA (100 micromol/L) perfused through the microdialysis probe evoked an increase in extracellular dopamine in the STN of the intact rat of about 170%. This coincided with significant increases in both extracellular glutamate (350%) and GABA (250%). The effect of NMDA perfusion on neurotransmitter release at the level of the STN was completely abolished by co-perfusion of the selective NMDA-receptor antagonist MK-801 (10 micromol/L), whereas subthalamic perfusion of MK-801 alone had no effect on extracellular neurotransmitter concentrations. Furthermore, NMDA induced increases in glutamate were abolished by both SCH23390 (8 micromol/L), a selective D1 antagonist, and remoxipride (4 micromol/L), a selective D2 antagonist. The NMDA induced increase in GABA was abolished by remoxipride but not by SCH23390. Perfusion of the STN with SCH23390 or remoxipride alone had no effect on extracellular neurotransmitter concentrations. The observed effects in intact animals depend on the nigral dopaminergic innervation, as dopamine denervation, by means of 6-hydroxydopamine lesioning of the substantia nigra, clearly abolished the effects of NMDA on neurotransmitter release at the level of the STN. Our work points to a complex interaction between dopamine, glutamate and GABA with a crucial role for dopamine at the level of the STN.

To broaden our knowledge about the central role of the angiotensin IV (Ang IV) peptide, we aimed to monitor its extracellular concentration in the brain using in vivo microdialysis. Ang IV was measured in the dialysates using a previously developed nano-LC-MS/MS assay with an LOD of 50 pM. Using this assay, baseline levels of Ang IV in dialysates from different brain structures were undetectable. However, immediately after microdialysis probe insertion, Ang IV could be detected in a concentration that varied between 120 and 187 pM. Using the zero-net-flux method, the extracellular levels of Ang IV in the striatum were estimated at 46 pM. These data may indicate that Ang IV is mainly present intracellularly. In addition, Ang IV was clearly measurable after striatal perfusion of Ang II. On the other hand, our nano-LC-MS/MS method was successful for the detection of Met-enkephalin and neurotensin in dialysates from the rat. In conclusion, the nano-LC-MS/MS method coupled with microdialysis is well suited to monitor the biologically significant conversion between Ang II and Ang IV in vivo, but physiological extracellular levels of Ang IV appear too low to be detected.

Locally administered angiotensin IV causes a dose-dependent increase of the dopamine levels in the striatum of the rat. The aminopeptidases insulin-regulated aminopeptidase (IRAP) and/or aminopeptidase N (AP-N) are proposed to be involved in this effect since both enzymes are inhibited by angiotensin IV. In agreement with this hypothesis we demonstrate that by using the AP-N selective inhibitor 7B, about 60% of the aminopeptidase activity in striatal membranes could be attributed to AP-N (pK(i)=9.20). Higher concentrations of 7B are capable of inhibiting IRAP as well (pK(i)=7.26). Interestingly, in vivo, inhibition of IRAP or AP-N activity does not appear to be involved in the angiotensin IV-mediated effect in the striatum since 7B itself is not capable to induce dopamine release such as observed with angiotensin IV. However, 7B at a concentration selective for inhibition of AP-N (100 nM) potentiates the angiotensin IV-mediated increase of dopamine, suggesting that inhibition of AP-N lengthens the half-life of angiotensin IV. On the other hand, inhibition of both AP-N and IRAP by perfusion of 500 nM 7B completely abolishes the effect of angiotensin IV. We therefore hypothesize that the effect of angiotensin IV on dopamine release in the striatum is mediated via activation of IRAP and/or AP-N, possibly acting as receptors for angiotensin IV.

A substantial amount of experimental evidence implicates neuropeptide Y (NPY) in the pathophysiology of epilepsy. Over the past 20 years, remarkable progress has been made in unraveling the mechanisms and receptors involved in the anticonvulsant effect of this abundantly expressed neuropeptide. Activation of Y(2) and/or Y(5) receptors and blockade of Y(1) receptors in the central nervous system suppresses seizures in a variety of animal seizure models. Orally available, brain penetrating Y(2) and/or Y(5) agonists, and possibly Y(1) antagonists, may therefore constitute a novel class of antiepileptic drugs, which could greatly benefit patients with medically refractory epilepsy. Significant progress has been made in identifying non-peptidergic Y(1) antagonists that fulfill these criteria, but suitable Y(2) and/or Y(5) agonists have proven to be more elusive. Innovative oral and parental drug delivery strategies which are currently under development may offer a means of using the more readily available peptidergic NPY receptor ligands in a clinical setting. Finally, gene therapy, antisense probes or RNA interference strategies which alter the expression of NPY or its receptors in specific brain regions may also be of use in the treatment of epilepsy, but will probably benefit a smaller subgroup of epilepsy patients, since they typically require an invasive procedure.

Quantifying low concentrations of neuropeptides in microdialysates requires a selective and sensitive analysis technique, such as nano-liquid chromatography/electrospray ionization tandem mass spectrometry (nanoLC/ESI-MS/MS). However, we observed reduced accuracy of the method due to matrix effects. Indeed, ESI-MS detection is known to be sensitive to matrix effects. Moreover, dialysates are complex mixtures of small molecules, peptides and other matrix compounds that can influence the ionization efficiency of the neuropeptide of interest and the stability of the peptide in the samples. In the study reported in this paper, we investigated whether the use of an internal standard (IS) can correct for these matrix effects. As a model compound for neuropeptides we used angiotensin IV (Ang IV). We compared the use of a structural analogue (norleucine1-Ang IV) with a stable isotope labeled (SIL) analogue. Linearity of the method was improved when either of the proposed ISs were applied. Only when using the SIL-IS could the repeatability of injection and the method's precision and accuracy be improved. Finally, the IS was able to correct for degradation of Ang IV in dialysates, prolonging the possible storage period of the samples. We conclude that the structural analogue is not suited as an IS and that the application of a SIL analogue is indispensable when quantifying Ang IV in dialysates using nanoLC/ESI-MS/MS detection., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Enhanced sensitivity is a well known benefit of miniaturised LC-electrospray (ESI)-MS/MS methods. The suitability of miniaturised LC-MS/MS for quantification of small molecules in dialysates was investigated using the anti-epileptic drug oxcarbazepine, its active metabolite, 10,11-dihydro-10-hydroxycarbamazepine, and the internal standard for microdialysis probe calibration, 2-methyl-5H-dibenz(b,f)azepine-5-carboxamide, as test compounds. ESI-MS detection is sensitive to matrix effects. Therefore, dialysate matrix effects were investigated by comparing the responses of standards made in water, Ringer's solution (salt solution used as perfusion fluid) and blank dialysate matrix. Due to the occurrence of ion suppression or enhancement, direct injection of dialysis samples onto the analytical column could not be applied for quantification of small molecules in dialysis samples. Column switching was necessary for desalting and preconcentration of the dialysates. However, this approach was not able to completely eliminate salt effects when the injection volume exceeded 1 microL. No differences in response between Ringer's solution and dialysate matrix were detected at capillary and nano-dimensions. Calibration standards should be prepared with Ringer's solution instead of water for quantitative analysis of microdialysates. A microbore, capillary and nano-LC-ESI-MS/MS method were compared in terms of method feasibility, linearity, sensitivity, accuracy and precision. Downscaling to capillary and nano-dimensions resulted in a gain in detection sensitivity of 5 and 50, respectively. Miniaturised LC-MS/MS was found to be fit for quantification of small molecules in dialysates with acceptable accuracy and method precision.

Due to its high affinity for [(125)I]Angiotensin IV, cystinyl aminopeptidase (CAP) has recently been assigned as the 'angiotensin AT(4) receptor'. Since the aminopeptidase N (AP-N) activity is also susceptible to inhibition by Angiotensin IV, it might represent an additional target for this peptide. Based on [(125)I]Angiotensin IV binding and catalytic activity measurements, we compared the ligand interaction properties of recombinant human CAP and human AP-N. Both enzymes displayed distinct pharmacological profiles. Although their activity is inhibited by Angiotensin IV and LVV-hemorphin 7, both peptides are more potent CAP-inhibitors. On the other hand, substance P and l-methionine have a higher potency for AP-N. High affinity binding of [(125)I]Angiotensin IV to CAP occurs in the presence of chelators but not to AP-N in either the absence or presence of chelators. These differences were exploited to determine whether CAP and/or AP-N are present in different cell lines (CHO-K1, COS-7, HEK293, SK-N-MC and MDBK). We provide evidence that CAP predominates in these cell lines and that, comparatively, CHO-K1 cells display the highest level of this enzyme.

Purpose: The substantia nigra pars reticulata (SNR) is known to play a role in gating and control of seizures. Prompted by the observation that intrahippocampal topiramate (TPM) administration does not suppress limbic seizures in the focal pilocarpine model, we investigated the role of the SNR in the anticonvulsant mechanism of action of TPM., Methods: Limbic seizures were evoked in freely moving rats by intrahippocampal administration of pilocarpine via a microdialysis probe. Changes in hippocampal extracellular (EC) glutamate and GABA concentrations were monitored. Effects of intraperitoneal (10-200 mg/kg), intrahippocampal (1-5 mM), and bilateral intranigral (100-300 nmol) TPM administration on pilocarpine-induced seizures and neurochemical changes were evaluated. Effects of TPM administration alone on hippocampal and nigral EC amino acid concentrations were also studied., Results: Systemic and intranigral, but not intrahippocampal TPM administration suppressed pilocarpine-induced seizures and neurochemical changes. Nigral GABA(A) receptor blockade by picrotoxin abolished the anticonvulsant effect of TPM in SNR. Systemic TPM administration increased hippocampal glutamate and decreased GABA. Intranigral TPM administration increased hippocampal glutamate, but not GABA. Intrahippocampal TPM increased hippocampal glutamate and GABA, but only at high concentrations., Conclusions: In the focal pilocarpine model, TPM does not exert its anticonvulsant effect at the site of seizure initiation. We identified the SNR as a site of action of TPM, and showed that the nigral GABA-ergic system is central to TPM's anticonvulsant effect in SNR. Anticonvulsant effects and neurochemical changes in hippocampus following intranigral TPM administration suggest the existence of a nigro-hippocampal circuit, which may be involved in the control of limbic seizures.

The anti-convulsant properties of angiotensin IV (Ang IV), an inhibitor of insulin-regulated aminopeptidase (IRAP) and somatostatin-14, a substrate of IRAP, were evaluated in the acute pilocarpine rat seizure model. Simultaneously, the neurochemical changes in the hippocampus were monitored using in vivo microdialysis. Intracerebroventricularly (i.c.v.) administered Ang IV or somatostatin-14 caused a significant increase in the hippocampal extracellular dopamine and serotonin levels and protected rats against pilocarpine-induced seizures. These effects of Ang IV were both blocked by concomitant i.c.v. administration of the somatostatin receptor-2 antagonist cyanamid 154806. These results reveal a possible role for dopamine and serotonin in the anti-convulsant effect of Ang IV and somatostatin-14. Our study suggests that the ability of Ang IV to inhibit pilocarpine-induced convulsions is dependent on somatostatin receptor-2 activation, and is possibly mediated via the inhibition of IRAP resulting in an elevated concentration of somatostatin-14 in the brain.

This paper provides an overview of the different classes of chiral selectors that are used in CE. The main properties of every class are described, together with the mechanism of enantioseparation. Newly introduced selectors are also discussed. Pharmaceutical and biomedical applications published from January 2004 till March 2005 are summarized.

Chiral separation of cetirizine, a second-generation H(1)-antagonist, was studied by CD-mediated CE. Several parameters, including pH, CD type, buffer concentration, type of co-ion, applied voltage and temperature, were investigated. The best conditions for chiral separation were obtained using a 75 mM triethanolamine-phosphate buffer (pH 2.5) containing 0.4 mg/mL heptakis(2,3-diacetyl-6-sulfato)-beta-CD and 10% ACN. Online UV detection was performed at 214 nm, a voltage of 20 kV was applied and the capillary was temperature controlled at 25 degrees C by liquid cooling. Hydrodynamic injection was performed for 1 s. The method was validated for the quantification of levocetirizine in tablets and for enantiomeric purity testing of the drug substance. Selectivity, linearity, LOD and LOQ, precision and accuracy were evaluated for both methods. The amount of levocetirizine dihydrochloride in the commercially available tablets was quantified and was found to be within the specification limits of the claimed amount (5 mg). The amount of distomer in levocetirizine drug substance was found to be 0.87 +/- 0.09% w/w, which is in agreement with the certificate of analysis supplied by the company.

A microbore LC-MS/MS method is developed and validated for the quantification of the anti-epileptic drug oxcarbazepine (OXC) and its active metabolite 10,11-dihydro-10-hydroxycarbamazepine (MHD) in rat brain microdialysates, together with the internal standard for microdialysis probe calibration, 2-methyl-5H-dibenz(b,f)azepine-5-carboxamide (m-CBZ). The benefits of gradient versus isocratic separation are shown, next to the improved sensitivity resulting from the addition of 0.1% formic acid to the mobile phase. The coupling of microdialysis with ESI-MS requires sample desalting for which column switching was applied. Using weighed regression to calculate the calibration curves (1-1000 ng/mL), the assay was validated in terms of linearity, accuracy and precision, yielding a sensitive (limit of quantification is 1 ng/mL) and selective method for quantification of OXC, MHD and m-CBZ. By applying this method, we were able to determine the extracellular concentrations of OXC and MHD during at least 4h after intraperitoneal (i.p.) administration of 10 mg/kg OXC.

Acute inflammation induces upregulation of IL-1beta both at the site of the peripheral inflammation and in the cerebrospinal fluid (CSF). The central increase of IL-1beta mainly contributes to the development of hypersensitivity. However, the spinal mechanisms for the effects of IL-1beta in nociceptive transmission are incompletely understood. It is also unknown whether previous sensitization changes IL-1beta activity. We therefore investigated the dose-effect relationship of intrathecal (i.t.) IL-1beta on spinal PGE(2) production in the absence and presence of peripheral formalin inflammation with spinal microdialysis in freely moving rats. The possible involvement of cyclooxygenase (COX) isoforms in the IL-1beta-mediated spinal PGE(2) production on the background of peripheral formalin inflammation was further evaluated with the selective COX-1 and COX-2 inhibitors. We found that the i.t. administration of IL-1beta, with doses of 1, 2, 8, or 16 ng, increased PGE(2) levels in CSF in a dose-related fashion. This IL-1beta-evoked PGE(2) release occurred within 30min after IL-1beta administration, peaked at 30-60 min interval, and returned gradually to the baseline level within 4h. Peripheral formalin inflammation in the paw induced a more prolonged effect of spinal IL-1beta with larger PGE(2) releases in the CSF compared with the non-inflammatory state, suggesting that peripheral inflammation enhances central sensitization. The COX-2 inhibitor SC58236 (15 mg/kg) reduced the IL-1beta-mediated PGE(2) increase in CSF by 86% while the COX-1 inhibitor SC58560 (15 mg/kg) had less effect (28%). Our study suggests that mainly the COX-2 enzyme mediates the IL-1beta-induced increase in spinal PGE(2) in the presence of peripheral formalin inflammation.

We recently showed that dopamine (DA) and serotonin (5-HT) exert anticonvulsant effects against limbic seizures in rats mediated by hippocampal D(2) and 5-HT(1A) receptor stimulation. For exogenously administered monoamines, anticonvulsant activity was only observed following 70--400% and 80--350% increases in baseline levels for dopamine and serotonin, respectively. The aim of the present microdialysis study was to investigate whether oxcarbazepine and its active metabolite, 10,11-dihydro-10-hydroxycarbamazepine (MHD) promote the release of hippocampal monoamines. Initially, concentration-response experiments were performed. Different concentrations of both compounds were perfused into the hippocampus via the microdialysis probe and tested for their effects on extracellular monoamine levels and anticonvulsant properties against pilocarpine-evoked seizures in rats. Anticonvulsant activity was always accompanied by significant increases in dopamine and serotonin levels. The anticonvulsant threshold concentrations for oxcarbazepine (100 microM) and 10,11-dihydro-10-hydroxycarbamazepine (250 microM) were associated with, respectively, 140 and 205% increases in hippocampal dopamine and 288 and 176% increases in serotonin concentrations. Co-perfusion of these anticonvulsant threshold concentrations for both compounds either with a selective D(2) or 5-HT(1A) antagonist abolished all anticonvulsant effects. This study shows that oxcarbazepine and 10,11-dihydro-10-hydroxycarbamazepine exert important monoamine promoting effects that, at least partly, contribute to the anticonvulsant mechanism of action of these compounds. The effects on dopamine and serotonin levels are therefore proposed as pharmacodynamic markers for the anticonvulsant activity of these compounds. These pharmacodynamic markers are here shown to be useful for the selection of anticonvulsant threshold concentrations of oxcarbazepine and 10,11-dihydro-10-hydroxycarbamazepine.

Previously we showed that treatment with mild hypothermia (34 degrees C for 2 h) after a focal cerebral infarct was neuroprotective by reducing apoptosis in the penumbra (cortex), but not in the core (striatum) of the infarct. In this study we examined whether administration of N-acetyl-aspartyl-glutamate (NAAG) in combination with mild hypothermia could improve striatal neuroprotection in the endothelin-1 rat model. NAAG (10 mg/kg i.p.) was injected under normothermic (37 degrees C) or mild hypothermic conditions, either 40 min before or 20 min after the insult. NAAG reduced caspase 3 immunoreactivity in the striatum, irrespective of the time of administration and brain temperature. This neuroprotective effect could be explained, at least partially, by decreased nitric oxide synthase activity in the striatum and was blocked by the group II metabotropic glutamate receptor antagonist, LY341495. Hypothermia applied together with NAAG reduced both cortical and striatal caspase 3 immunoreactivity, as well as the overall ischaemic damage in these areas. However, no pronounced improvement was seen in total damaged brain volume. Extracellular glutamate levels did not correlate with the observed protection, whatever treatment protocol was applied. We conclude that treatment with NAAG causes the same degree of neuroprotection as treatment with hypothermia. Combination of the two treatments, although reducing apoptosis, does not considerably improve ischaemic damage.

The purpose of the present study was to examine the effects of an acute dose of the dual dopamine (DA) and norepinephrine (NE) reuptake inhibitor bupropion (Bup) on brain (T(brain)), body core (T(core)), and tail skin (T(tail)) temperature in freely moving rats and to simultaneously monitor the extracellular neurotransmitter concentrations in the preoptic area and anterior hypothalamus (PO/AH). A microdialysis probe was inserted in the PO/AH, and samples for NE, DA, and serotonin (5-HT) were collected every 20 min before and after the injection of 17 mg/kg of Bup, for a total sampling time of 180 min. T(core) was monitored using a biotelemetry system. T(brain) and T(tail), an index of heat loss response, were also measured. Both NE and DA levels in the PO/AH significantly increased after Bup injection compared with the baseline levels, reaching approximately 450 and 230%, respectively, 40 min after injection. There was no effect on 5-HT release. The neurotransmitter changes were accompanied by a significant decrease in T(tail) and an increase in both T(brain) and T(core) compared with the baseline levels. The present results demonstrate that inhibition of NE and DA reuptake suppresses heat loss mechanisms and elevates T(brain) and T(core) in freely moving rats.

Previously, we showed that treatment with resuscitative, post-ischaemic mild hypothermia (34 degrees C for 2 h) reduced apoptosis in the penumbra (cortex), but not in the core (striatum) of an endothelin-1 (Et-1)-induced focal cerebral infarct in the anaesthetized rat. Therefore, the purpose of this study was to investigate by which pathways resuscitative mild hypothermia exerts its neuroprotective effect in this model. The amino acids glutamate, serine, glutamine, alanine, taurine, arginine and the NO-related compound citrulline were sampled from the striatum and cortex of the ischaemic hemisphere using in vivo microdialysis. The in vivo salicylate trapping method was applied for monitoring hydroxyl radical formation via 2,3 dihydroxybenzoic acid (2,3 DHBA) detection. Caspase-3, neuronal nitric oxide synthase (nNOS) immunoreactivity and the volume of ischaemic damage were determined 24 h after the insult. In both the striatum and the cortex, Et-1-induced increases in glutamate, taurine and alanine were refractory to mild hypothermia. However, mild hypothermia significantly attenuated the ischaemia-induced 2,3 DHBA levels and the nNOS immunoreactivity in the cortex, but not in the striatum. These observations were associated with a decreased caspase-3 immunoreactivity. These results suggest that mild hypothermia exerts its neuroprotective effect in the penumbra partially by reducing nNOS activity and thereby preventing oxidative stress. Furthermore, we confirm our previous findings that the neuroprotective effect of resuscitative hypothermia is not mediated by changes in ischaemia-induced amino acid release as they could not be associated with the ischaemia-induced damage in the Et-1 rat model.

Cystinyl aminopeptidase has one Zn2+-binding motif and is a member of the M1 aminopeptidase family. Ion modulation of its catalytic activity was studied in membranes of CHO-K1 cells (Chinese-hamster ovary K1 cells) using L-leucine-p-nitroanilide as substrate. The planar bidentate chelators 1,10-phenanthroline and 2,2'-bipyridine inhibited the activity in a concentration-dependent manner with Hill slopes of 3.32+/-1.78 and 2.10+/-0.26 respectively. The acetic acid-containing chelators EDTA, EGTA and DTPA (diethylenetriamine-N,N,N',N'',N''-penta-acetic acid) weakly affected the activity, but they increased the potency of the planar chelators up to a limit, at which Hill slopes became close to unity. Moreover, competition between 1,10-phenanthroline and the substrate only took place in the presence of EDTA. These findings are compatible with a model in which the bidentate chelators inhibit enzyme activity by decreasing the free Zn2+ concentration. By removing a modulatory ion from an allosteric site at the enzyme, the acetic acid-containing chelators facilitate the direct interaction between the bidentate chelators and the catalytic Zn2+. The inhibitory effect of EDTA plus 1,10-phenanthroline could be completely reversed by Zn2+. Ca2+ and Mg2+ increased the potency of Zn2+ for this process. This is expected if they interact with the modulatory site to decrease the sensitivity of the enzyme towards 1,10-phenanthroline. Conversely, the bidendate chelators increased the high-affinity [125I]angiotensin IV binding to the membranes and this was potentiated by the acetic acid-containing chelators. These findings support the concept that high-affinity [125I]angiotensin IV binding, previously referred to as 'AT4 receptor binding', only occurs for the cystinyl aminopeptidase apoenzyme.

The effect of angiotensin (Ang)-1-7 on dopamine, gamma-aminobutyric acid (GABA) and glutamate release in the striatum of the rat was examined using in vivo microdialysis. Ang-(1-7) was administered locally in the striatum through the microdialysis probe. At a concentration of 100 microm, Ang-(1-7) caused a significant increase in extracellular dopamine and GABA but had no effect on glutamate release. The Ang-(1-7)-induced dopamine release was blocked by EC33, an inhibitor of aminopeptidase A, an enzyme which converts Ang-(1-7) into Ang-(3-7), suggesting that this effect occurs after metabolism into Ang-(3-7). Indeed, administration of Ang-(3-7) (10-100 microm) into the striatum caused a more potent increase in the striatal dopamine release than Ang-(1-7). Because Ang-(3-7) is an inhibitor of insulin-regulated aminopeptidase (IRAP) and because Ang IV, another IRAP inhibitor, also causes a concentration-dependent increase in dopamine in the rat striatum, IRAP may be involved in this effect. In contrast, EC33 had no effect on the Ang-(1-7)-induced GABA increase but the GABA release was blocked by the putative AT(1-7) receptor antagonist A779 (0.1 microm) and by the nitric oxide synthase inhibitor L-NAME (1 mm). These drugs could not block the effect of Ang-(1-7) on the striatal dopamine release suggesting that only the observed effects on GABA release are mediated by the AT(1-7) receptor and/or are associated with a release of nitric oxide.

Various antiepileptic drugs were shown to be substrates for multidrug transporters at the level of the blood-brain barrier. These ATP-dependent efflux pumps actively limit brain accumulation of xenobiotics and drugs. Intrahippocampal oxcarbazepine perfusion in rat was previously shown to exert anticonvulsant effects associated with increases in extracellular dopamine and serotonin levels. In contrast, preliminary studies in our laboratory revealed that no anticonvulsant or monoaminergic effects could be obtained after systemic oxcarbazepine administration. The present in vivo microdialysis study was conducted to investigate the impact of the transport kinetics of oxcarbazepine across the blood-brain barrier on the observed treatment refractoriness. More precisely, the influence of intrahippocampal perfusion of verapamil, a P-glycoprotein inhibitor, and probenecid, a multidrug resistance protein inhibitor, on the blood-brain barrier passage and anticonvulsant properties of oxcarbazepine were investigated in the focal pilocarpine model for limbic seizures. Simultaneously, the effects on hippocampal monoamines were studied as pharmacodynamic markers for the anticonvulsant activity. Although systemic oxcarbazepine administration alone failed in preventing the animals from developing seizures, coadministration with verapamil or probenecid offered complete protection. Concomitantly, significant increases in extracellular hippocampal dopamine and serotonin levels were observed within our previously defined anticonvulsant monoamine range. The present data indicate that oxcarbazepine is a substrate for multidrug transporters at the blood-brain barrier. Coadministration with multidrug transporter inhibitors significantly potentiates the anticonvulsant activity of oxcarbazepine and offers opportunities for treatment of pharmacoresistant epilepsy.

Purpose: Our recent work (Clinckers et al., J Neurochem 2004;89:834-43) demonstrated that intrahippocampal perfusion of 2 nM dopamine or serotonin via a microdialysis probe offered complete protection against focal pilocarpine-induced limbic seizures and did not influence basal extracellular hippocampal glutamate levels. Ten nanomolar dopamine or serotonin perfusion, however, worsened seizures and was accompanied by significant extracellular glutamate increases to approximately 200%. The significance of these glutamate elevations in seizure generation remains unclear. The present microdialysis study investigated the modulatory role of extracellular hippocampal glutamate levels in these monoaminergic protective and proconvulsant effects., Methods: A first group of male Wistar albino rats was perfused intrahippocampally for 240 min with 6.25 microM glutamate alone to increase extracellular levels by 200%. Other animals were perfused with anticonvulsant concentrations of monoamines throughout the experiments while receiving continuous coperfusions of 6.25 microM glutamate either before, during, and after (240 min) or only after (100 min) pilocarpine perfusion (40 min). Rats were scored for epileptic behavior, and the mean scores were compared with those of the control group. Microdialysates were analyzed for monoamine and glutamate content with microbore liquid chromatography., Results: No convulsions occurred during glutamate perfusion alone. When monoamines and glutamate were coperfused before pilocarpine administration, the anticonvulsant effect of the monoamines was lost. Glutamate addition after pilocarpine administration did not affect monoaminergic seizure protection., Conclusions: These results indicate that extracellular glutamate increases per se do not necessarily induce seizures but that they can modulate the anticonvulsant effects exerted by hippocampal monoamines.

We compared the effect of an injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle (MFB) and into the striatum on different parameters for evaluation of motor dysfunction and dopamine denervation in rats, as a function of time. A combination of behavioural, neurochemical and histological techniques was employed. Amphetamine-induced rotation is shown to provide a first rough estimation of motor impairment. Indeed, the number of rotations observed after amphetamine administration can distinguish between a partial and a near complete (>90%) denervation in the substantia nigra. However, lesion sizes of 50-80% resulted in similar rotational behaviour. Similarly, the elevated body swing test (EBST) can determine severe lesions, but is not sensitive enough in the partial model. In both models, determination of the dopamine tissue content with HPLC is a more precise measure of striatal dopamine innervation than striatal TH-immunostaining. The number of cells estimated by TH- and Nissl-staining correlated well in the striatal model, but there was a discrepancy between both measures in the MFB-lesioned animals. Therefore, additional Nissl-staining is necessary for better estimation of the size of the lesion at the level of the substantia nigra or ventral tegmental area in the severely lesioned animals. The MFB lesion model mimics end-stage Parkinson's disease. The striatal injection of 6-OHDA described here cannot be considered a progressive model, since there was no change in the number of TH-immunoreactive cells in the substantia nigra up to 8 weeks post-lesioning. However, the partial denervation renders its quite suitable for mimicking early stage Parkinson's disease, and is thus suitable for testing possible neuroprotective and neurotrophic drugs.

Microdialysis coupled to liquid chromatography (LC) has proven to be a valuable in vivo sampling technique for studying neurotransmitter changes in normal and ischaemic brain. However, few analytical methods have described the simultaneous determination of amino acids, relevant in stroke research, together with the nitric-oxide-related compound citrulline. Therefore, we developed a gradient LC method for the quantitative simultaneous determination of aspartate, glutamate, serine, glutamine, arginine, taurine, alanine and citrulline in dialysates of rat brain using narrowbore LC with o-phthalaldehyde-2-mercaptoethanol pre-column derivatisation and fluorescence detection. The proposed method is a thoroughly validated, fully automated and robust LC method for the determination of amino acids in a wide concentration range. The method was applied for the determination of amino acids and the citrulline/arginine ratio in the Et-1 model for focal cerebral ischaemia.

Objective: To study the effects of mild hypothermia on the 24h concentration of serum astroglial of S-100 beta protein in patients who survived cardiac arrest (CA)., Design: A prospective, randomised, clinical study in a university teaching hospital., Patients: Sixty-one resuscitated patients were randomised into two prospective studies, known as the short study period (SSP) (n = 33 patients) and the long study period (LSP) (n = 28 patients). In the SSP study, patients older than 18 years of age and surviving asystole or pulseless electrical activity were included. In the LSP study, patients with ventricular fibrillation (VF) or non-perfusing ventricular tachycardia (VT) aged between 18 and 75 years were included. In each of the study groups, the patients were further randomised into either normothermic or hypothermic subgroups. The standard supportive therapy was similar, only the devices used to reduce the body temperature and the period of hypothermia were different. Serum samples for the measurement of astroglial S-100 beta protein were collected at admission and 24h later., Results: During the first 24h after the cardiac arrest, the concentration of astroglial serum S-100 beta protein decreased significantly in the hypothermic cohort. In the normothermic cohort, the decrease of serum astroglial S-100 beta protein was less pronounced and even increased in the normothermic LSP group., Conclusion: Induced mild hypothermia reduced the 24h astroglial serum S-100 beta protein concentration and might play a neuroprotective effect after cardiac arrest.

We recently showed that intrahippocampally administered dopamine and serotonin exert concentration-dependent non-protective, protective and proconvulsant effects against limbic seizures in rats. Anticonvulsant action was mediated via, respectively, hippocampal D2 and 5-HT1A receptor stimulation, while proconvulsant effects were associated with concomitant hippocampal glutamate increases. We here examined whether increases in endogenous hippocampal dopamine and serotonin exert similar actions. Initially, dose-response experiments were performed with intrahippocampal perfusions of GBR-12909 and citalopram, respectively, selective dopamine and serotonin re-uptake blockers. Based on their effects on monoaminergic release, a potential non-protective, protective and proconvulsant concentration was selected. The predicted non-protective GBR-12909 (10 microM) and citalopram (0.5 microM) concentrations failed to prevent pilocarpine-induced seizures. The predicted protective GBR-12909 (100 microM) and citalopram (1 microM) perfusions resulted in complete anticonvulsant action, again mediated by D2 and 5-HT1A receptors. Unexpectedly, at predicted proconvulsant concentrations complete anticonvulsant action was obtained and hippocampal Glu remained unaltered. This study shows that selective monoamine re-uptake blockers have important anticonvulsant properties. Based on the previously established anticonvulsant monoamine ranges, anticonvulsant threshold concentrations can be predicted for compounds with endogenous dopamine or serotonin promoting effects. Non-selective actions curtailing glutamatergic activity may further be responsible for the unexpected anticonvulsant effects at predicted proconvulsant concentrations.

The separation and simultaneous enantiomeric separation of three neutral 1,4-dihydropyridine (DHP) derivatives (nimodipine, nisoldipine and nitrendipine) was studied using electrokinetic chromatography. Bile salts allowed the non-chiral separation of these DHP derivatives. With the taurine-conjugated bile salts a beginning of enantiomeric separation was observed for nimodipine and nisoldipine. Achiral micelles of sodium dodecyl sulphate mixed with neutral cyclodextrins did not allow enantioseparation. Baseline chiral separation of nisoldipine and nimodipine was obtained with carboxymethyl-beta-cyclodextrin at pH 5.0. The buffer type affected the chiral separation, especially in the case of nisoldipine. The addition of organic solvent decreased the enantioresolution of nimodipine. However, the resolution between the nisoldipine enantiomers was increased when methanol or ethanol were added to the background electrolyte. Varying the temperature had almost no effect on the enantioresolution of nisoldipine, whereas with nimodipine a clear improvement at lower temperatures was observed. Using the optimised method, the selectivity of this method was investigated for three possible impurities of nisoldipine.

We investigated the possible neuroprotective effect of the dopamine (DA) receptor agonist R-apomorphine (R-APO) within the striatal 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. In one group of rats, R-APO administration (10 mg/kg/day, s.c.) started 15 min before 6-OHDA-injection. In a second group, R-APO administration started 24 h after lesion induction. Both groups received R-APO chronically for 11 days. Testing was carried out 2 weeks post-lesioning. R-APO treatment, whether started before or after the lesion induction, significantly reduced both the amphetamine-induced ipsiversive rotation and the size of the lesion at the level of the substantia nigra. Moreover, the dopamine cell shape and size resembled that observed in intact animals. R-APO treatment had no effect on the number of cells in the substantia nigra of intact rats, but significantly increased the number of cells in the ventral tegmental area (VTA), suggesting selective neurotrophic properties of R-APO in this region. R-APO treatment significantly attenuated the 6-OHDA-induced striatal DA depletion and DOPAC/DA ratios were normalized. Finally, an acute injection of 10 mg/kg R-APO was unable to scavenge 6-OHDA or MPP(+)-induced hydroxyl radicals as determined with the in vivo salicylate trapping technique. These data provide further evidence of the neurorescuing properties of R-APO. At least at the dose used in this study, this effect possibly occurs via mechanisms other than scavenging of hydroxyl radicals. In intact rats, we also show neurotrophic effects of the R-APO treatment. These seem to be limited to the VTA.

The present study investigated whether postischemic mild hypothermia attenuates the ischemia-induced striatal glutamate (GLU) and dopamine (DA) release, as well as astroglial cell proliferation in the brain. Anesthetized rats were exposed to 8 min of asphyxiation, including 5 min of cardiac arrest. The cardiac arrest was reversed to restoration of spontaneous circulation (ROSC), by brief external heart massage and ventilation within a period of 2 min. After the insult and during reperfusion, the extracellular glutamate and dopamine overflow increased to, respectively, 3000% and 5000% compared with the baseline values in the normothermic group and resulted in brain damage, ischemic neurons and gliosis. However, when hypothermia was induced for a period of 60 min after the insult and restoration of spontaneous circulation, the glutamate and dopamine overflows were not significantly different from that in the sham group. Histological analysis of the brain showed that postischemic mild hypothermia reduced brain damage, ischemic neurons, as well as astroglial cell proliferation. Thus, postischemic mild hypothermia reduces the excitotoxic process, brain damage, as well as astroglial cell proliferation during reperfusion. Moreover, these results emphasize the trigger effect of dopamine on the excitotoxic pathway.

The effect of angiotensin (Ang) IV, an inhibitor of insulin-regulated aminopeptidase (IRAP), on extracellular dopamine levels in the striatum of freely moving rats was examined using in vivo microdialysis. The Ang IV was administered locally in the striatum through the microdialysis probe. A concentration-dependent (10-100 microm) increase in extracellular striatal dopamine was observed. The effect of Ang II (10-100 microm), which has only a weak affinity for IRAP, was similar to that observed for Ang IV. The effects of both peptides could not be blocked by the AT1 antagonist candesartan (10 nm and 1 microm) nor by the AT2 antagonist S-(+)-1-([4-(dimethylamino)-3-methylphenyl]methyl)-5-(diphenyl-acetyl)-4,5,6,7-tetrahydro-1H-amidazo(4,5-c) pyridine-6-carboxylic acid (1 microm), suggesting that the observed effects are both AT1 and AT2 independent. The effect of Ang II could be blocked by the aminopeptidase-A inhibitor (S)-3-amino-4-mercaptobutylsulphonic acid as well as the aminopeptidase-N inhibitor 2-amino-4-methylsulphonylbutane thiol, indicating that the effect of Ang II is mediated via metabolism into Ang IV. Other IRAP inhibitors, such as Divalinal-Ang IV and LVV-haemorphin-7, had similar effects on extracellular dopamine levels as compared with Ang IV. We propose a role for IRAP as mediator for the effects of Ang IV and related peptides on extracellular dopamine levels in the striatum of the rat., (Copyright 2004 International Society for Neurochemistry)

The angiotensin II C-terminal hexapeptide fragment angiotensin IV (Ang IV) exerts central and cardiovascular effects. Cystinyl aminopeptidase (EC 3.4.11.3), a membrane-associated zinc-dependent metallopeptidase of the M1 family, has recently been found to display high affinity for Ang IV and it was proposed to represent the AT4 receptor. We present evidence for the presence of endogenous cystinyl aminopeptidase in membranes from Chinese hamster ovary (CHO-K1) cells by binding studies with [125I]Ang IV and by measuring the cleavage of L-leucine-p-nitroanilide. The equilibrium dissociation constant of [125I]Ang IV in saturation binding studies (KD= 0.90 nM) was similar to the value (KD= 0.70 nM) calculated from the association and dissociation rates. Binding was displaced with high potency by the "AT4 receptor" ligands (Ang IV > divalinal1-Ang IV approximately LVV-hemorphin-7 approximately LVV-hemorphin-6 > Ang (3-7) > Ang III > Ang (4-8)) but not by AT1/AT2 receptor antagonists. Enzymatic activity in CHO-K1 cell membranes was competitively inhibited upto 94% by Ang IV and other "AT4 receptor" ligands (Ang IV > Ang III approximately divalinal1-Ang IV approximately Ang (3-7) approximately LVV-hemorphin-7 > Ang (4-8) approximately LVV-hemorphin-6). High affinity binding of [125I]Ang IV required the presence of metal chelators and the ligands such as Ang IV and LVV-hemorphin-7 displayed higher potency in the binding studies as in the enzyme assay. This difference in potency varied from one peptide to another. These pharmacological properties match those previously reported for the recombinantly-expressed human cystinyl aminopeptidase in embryonal kidney cells.